Gain-of-function ADCY5 mutations in familial dyskinesia with facial myokymia

Abstract

Objective: To identify the cause of childhood onset involuntary paroxysmal choreiform and dystonic movements in 2 unrelated sporadic cases and to investigate the functional effect of missense mutations in adenylyl cyclase 5 (ADCY5) in sporadic and inherited cases of autosomal dominant familial dyskinesia with facial myokymia (FDFM).

Methods: Whole exome sequencing was performed on 2 parent-child trios. The effect of mutations in ADCY5 was studied by measurement of cyclic adenosine monophosphate (cAMP) accumulation under stimulatory and inhibitory conditions.

Results: The same de novo mutation (c.1252C>T, p.R418W) in ADCY5 was found in both studied cases. An inherited missense mutation (c.2176G>A, p.A726T) in ADCY5 was previously reported in a family with FDFM. The significant phenotypic overlap with FDFM was recognized in both cases only after discovery of the molecular link. The inherited mutation in the FDFM family and the recurrent de novo mutation affect residues in different protein domains, the first cytoplasmic domain and the first membrane-spanning domain, respectively. Functional studies revealed a statistically significant increase in β-receptor agonist-stimulated intracellular cAMP consistent with an increase in adenylyl cyclase activity for both mutants relative to wild-type protein, indicative of a gain-of-function effect.

Interpretation: FDFM is likely caused by gain-of-function mutations in different domains of ADCY5-the first definitive link between adenylyl cyclase mutation and human disease. We have illustrated the power of hypothesis-free exome sequencing in establishing diagnoses in rare disorders with complex and variable phenotype. Mutations in ADCY5 should be considered in patients with undiagnosed complex movement disorders even in the absence of a family history.

FIGURE 1

Schematic of ADCY5 showing the protein domains and locations of the mutations. The protein contains two 6-helical section membrane-spanning domains, M1 and M2, and 2 bipartite cytoplasmic domains, C1 and C2, which when brought together form a catalytic pocket for conversion of adenosine triphosphate (ATP) to cyclic adenosine-3′,5′-monophosphate (cAMP). Brackets denote synthetic reagents used. Fsk=forskolin, a receptor-independent stimulator of most adenylyl cyclases; Isop=isoproterenol, a β-adrenergic receptor (AR) agonist; Prop=propranolol, a β-AR antagonist.

FIGURE 2

Assessment of effect of ADCY5 mutations on protein expression in HEK293 cells transfected with ADCY5 expression constructs. HEK293 cells were transfected with plasmid DNA of enhanced green fluorescence protein (EGFP) vector alone, EGFP-tagged wild-type (ADCY5-WT), mutant ADCY5–726T, or mutant ADCY5–418W. Twenty-four hours later, cells were treated with carrier alone, forskolin (Fsk), isoproterenol (Isop), or propranolol (Prop) plus isoproterenol for 15 minutes. Cells were then harvested, and protein expression was measured by immunoblotting with anti-ADCY5. (A) ADCY5 immunoblot. Two bands at ∼130kDa and ∼160kDa were detected, corresponding to endogenous ADCY5 and transfected ADCY5 fused with the EGFP tag (ADCY5-EGFP), respectively. (B) Analysis of ADCY5 expression levels. Expression levels of ADCY5 were quantified and normalized with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels and standardized to expression in cells transfected with ADCY5-WT with basal carrier treatment. Error bars are standard errors from triplicate determinations. Student t test showed that there was no significant difference (p>0.05) between wild-type and mutant ADCY5 constructs for any of the conditions.

FIGURE 3

Intracellular cyclic adenosine-3′,5′-monophosphate (cAMP) assessed by enzyme immunoassay. HEK293 cells were transfected with empty enhanced green fluorescence protein (EGFP) vector (Vector) or expression constructs of EGFP tagged wild type (WT)-, 418W- or 726T–ADCY5 for 24 hours. Cells were treated under different adenylyl cyclase enzyme activating conditions—carrier alone, isoproterenol (Isop), or propranolol (Prop) plus Isop for 15 minutes—and cAMP level was assessed using an enzyme immunoassay. Treatment with Isop resulted in markedly increased cAMP from the carrier-treated basal levels. In comparison to ADCY5-WT, both mutant constructs demonstrated significantly increased levels of intracellular cAMP. Pretreatment with Prop abrogated the stimulatory effect of Isop for all constructs. Data shown are averages of 3 independent replicates standardized to activity of the empty vector under carrier-treated basal conditions. This value is designated as 1 unit on the y-axis. Statistical analyses were performed using Student t test; significant p values are indicated in the figure. Error bars indicate standard error of the mean.